The Development And Application Of Optical Biosensor Based On Aptamer For Salmonella Determination

Salmonella is a pathogenic microorganism,it can infect humans through environmental mediations such as gastroenteritis,bacteremia and sepsis,typhoid,and intestinal fever.Among them,S.Typhimurium is the most serious disease that can be fatal.The incubation period is1¹4 days,and it seriously harms human health.Salmonella is usually transmitted by polluted water systems such as discharged sewage and faeces of animals.Once humans drink water containing Salmonella,they will cause serious harm to the human health.In recent years,the detection of Salmonella in water has attracted attention around the world,and various testing standards have been issued.However,the traditional detection methods of pathogenic microorganisms still have some limitations.Therefore,the rapid and accurate of S.Typhimurium detection is of great significance for environmental safety testing and disease diagnosis.In this dissertation,three biosensor for detecting S.Typhimurium in water was constructed using optical sensing and specific recognition of aptamers.The main contents of the research paper are summarized as follows:Firstly,a novel fluorescence biosensing strategy for ultrasensitive and specific detection of S.Typhimurium based on target-triggered enzymatic repairing amplification?ERA?has been developed.This strategy relies on target-aptamer binding mediated ERA reaction,which is carried out cyclically with the help of polymerase and two DNA repairing enzymes,uracil-DNA glycosylase?UDG?and endonuclease IV?Endo IV?to produce amplified fluorescence signal.In our assay,the specially designed hairpin probe?HAP?is used as DNA template responsible for producing a great quantity of reporter oligonucleotides and secondary primers,which can initiate a new cycle of polymerization-repairing amplification.Moreover,by the combination of polymerase-catalyzed incorporation of lesion bases with UDG and Endo IV-assisted ERA,multiple cycle of amplification of the recognition event is achieved,enabling ultrasensitive detection of pathogenic bacteria.Under optimal conditions,this biosensor exhibits ultrasensitivity toward target pathogenic bacteria with detection limits of 10 cfu·mL^-1and a detection range of 5 orders of magnitude.Additionally,the biosensor has the ability of combating nonspecific background.Furthermore,an archer probe containing the anti-target aptamer sequence and a primer sequence is designed,which translates the binding of target to aptamer into the presence of primer sequence,enabling the detection of various targets,such as protein,DNA,small molecular,and any substance possessing its aptamer.Hence,the proposed target-triggered ERA-based signal-on fluorescence sensing strategy indeed create a versatile and useful platform for detection of pathogenic bacteria,related food safety analysis and clinical diagnosis.Next,we describe a fluorometric strategy for the detection of S.Typhimurium detection with ultrasensitivity and high specificity is reported.This strategy relies on the combination of target-modulated photoinduced electron transfer?PET?between G-quadruplex DNAzyme and DNA?labeled with silver nanoclusters?along with hairpin probe-based circular exponential amplification.The reaction system involves three hairpin probes?H1,H2 and H3?.Probe H1contains an aptamer against S.Typhimurium and the recognition sequence for nicking endonuclease.It is used to recognize S.Typhimurium and participates in polymerase-catalyzed target recycle amplification and secondary-target recycle amplification.Probe H2 contains an aptamer against hemin and is used to form the G-quadruplex DNAzyme in the presence of hemin and potassium ion.It acts as the electron acceptor and quenches the fluorescence of the AgNC labeled DNA.Fluorescence is best measured at excitation/emission wavelengths of567 nm/650 nm.Probe H3 contains the template sequence for the synthesis of AgNCs and the H2-annealing sequence.Both H2 and H3 are utilized to perform a strand displacement reaction and to achieve PET between G-quadruplex DNAzyme and DNA/AgNCs.To the best of our knowledge,this is the first example of a PET between G-quadruplex DNAzyme and DNA/AgNCs coupled with circular exponential amplification.The assay has an ultra-low detection limit 8 cfu·mL^-1 of S.Typhimurium.The assay is rapid,accurate,inexpensive and simple.Hence,the strategy may represent a useful platform for ultrasensitive and highly specific detection of pathogenic bacteria as encountered in food analysis and clinical diagnosis.Finally,we developed a biosensor based on G-quadruplex DNase-mediated plasmon resonance coupling and surface-enhanced Raman scattering?SERS?signal transduction to detect S.Typhimurium.In this method,an arched probe was designed to identify the target,and two hairpin probes?H1 and H2?were used in the amplification of the hybridization chain reaction to generate a large amount of G-quadruplex DNase,and functionalized gold nanoparticles was prepared.L-Cysteine can induce aggregation of gold nanoparticles by hydrogen bonding and electrostatic interactions with gold nanoparticles,while G-quadruplex DNase catalyzes the conversion of L-cysteine to cystine,inhibiting the aggregation of gold nanoparticles to control SERS.Compared with the traditional methods,it is simple,rapid,and stable.It does not require any biomolecule labeling and tedious elution steps.Hence,the strategy may represent a useful platform for ultrasensitive and highly specific detection of S.Typhimurium.